High Dose of Antithrombin Suppresses Neutrophil Extracellular Trap Formation in Human Neutrophils In Vitro Following Lipopolysaccharide- and Platelet-Induced Stimulation

Abstract

Objective:

During sepsis, blood antithrombin (AT) concentrations decrease; administering AT to patients with sepsis-associated intravascular coagulation has been shown to improve their prognosis. Neutrophil extracellular traps (NETs) are closely related to immunothrombosis; however, the relation between AT and NETs remains nebulous. This study examined the effects of AT on NETs of human neutrophils in vitro.

Methods:

Blood samples were collected from healthy volunteers, and the isolated neutrophils were seeded onto collagen-coated cell culture plates. The cells were then stimulated by lipopolysaccharide (LPS) with platelets. Human serum albumin (hAlb) was used as a control for AT. AT (0.01–10 U/mL) or hAlb was added 30 minutes after LPS and platelet treatment. The timing of AT addition was determined by the results of a single cell NETs assay. NET formation was assessed using enzyme-linked immunosorbent assay and immunostaining. Peptidyl arginine deiminase 4 (PAD4) nuclear transfer was detected by Western blotting.

Results:

Lipopolysaccharide-induced deoxyribonucleic acid (DNA)-binding neutrophil elastase substantially. NET formation over time was 3%, 15%, 50%, and 70% of the total neutrophils at 30, 60, 100, and 120 minutes after LPS stimulation, respectively. NETs evaluation in addition to AT at 15, 30, or 60 minutes after LPS stimulation showed that AT inhibited NET formation only at 30 minutes. High AT concentrations (i.e., 2–10 U/mL) substantially decreased NET formation and decreased nuclear expression of PAD4.

Conclusion:

The high concentrations of AT decrease NETs in human neutrophils induced by LPS and platelets via PAD4 pathway.